Supplementary data

S1: Absence of leak of the CMV-loxP-CAT-loxP-LacZ construct in absence of exogenous Cre recombinase activity. ß-galactosidase activity was measured in native RCCD2 cells transiently transfected with 1µg of CMV-loxP-CAT-loxP-LacZ (3) and compared with untransfected RCCD2 cells (1). A negative control was performed using 1µg of CMV-loxP-CAT-loxP-eGFP-hMR, which does not encode for the LacZ gene (2). A positive transient transfection control was performed using 0.5µg of the SV40-ßGal vector (4). Results indicated that there is no read through transcription of LacZ in the presence of the floxed cassette loxP-CAT-loxP-LacZ and in native RCCD2 cells, i.e. in absence of exogenous Cre recombinase. Values are means ± SEM (n=3). S2: Specificity of the transactivation properties of the eGFP-hMR expressed in mIN 8.11 eGFP-hMR cells. Experiments were performed on cells cultured in steroid-free medium. Transcriptional activity of the eGFP-hMR protein was tested on the MMTV-luciferase reporter plasmid and on the promoter-less luciferase plasmid pGL2-Basic(Promega), in the presence or absence of 10nM 4-OH-Tam, and 1nM aldosterone. In the presence or absence of eGFP-hMR expression (in cells pre-treated or not with 4-OH-Tam), aldosterone has no detectable effect of on the promoter-less pGL2-Basic vector. By contrast, transcriptional activity of the MMTV promoter is observed in the presence of aldosterone when the eGFP-hMR is expressed (in cells pre-treated with 4-OH-Tam). Of note, 4-OH Tam per se has no effect on MMTV transcriptional activity, i.e. in cells treated or not with 4-OH Tam, in the absence of aldosterone. Values are means ± SEM (n=3).

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• Figure and Figure legends - S1: Absence of leak of the CMV-loxP-CAT-loxP-LacZ construct in absence of exogenous Cre recombinase activity. ß-galactosidase activity was measured in native RCCD2 cells transiently transfected with 1µg of CMV-loxP-CAT-loxP-LacZ (3) and compared with untransfected RCCD2 cells (1). A negative control was performed using 1µg of CMV-loxP-CAT-loxP-eGFP-hMR, which does not encode for the LacZ gene (2). A positive transient transfection control was performed using 0.5µg of the SV40-ßGal vector (4). Results indicated that there is no read through transcription of LacZ in the presence of the floxed cassette loxP-CAT-loxP-LacZ and in native RCCD2 cells, i.e. in absence of exogenous Cre recombinase. Values are means ± SEM (n=3). S2: Specificity of the transactivation properties of the eGFP-hMR expressed in mIN 8.11 eGFP-hMR cells. Experiments were performed on cells cultured in steroid-free medium. Transcriptional activity of the eGFP-hMR protein was tested on the MMTV-luciferase reporter plasmid and on the promoter-less luciferase plasmid pGL2-Basic(Promega), in the presence or absence of 10nM 4-OH-Tam, and 1nM aldosterone. In the presence or absence of eGFP-hMR expression (in cells pre-treated or not with 4-OH-Tam), aldosterone has no detectable effect of on the promoter-less pGL2-Basic vector. By contrast, transcriptional activity of the MMTV promoter is observed in the presence of aldosterone when the eGFP-hMR is expressed (in cells pre-treated with 4-OH-Tam). Of note, 4-OH Tam per se has no effect on MMTV transcriptional activity, i.e. in cells treated or not with 4-OH Tam, in the absence of aldosterone. Values are means ± SEM (n=3).